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Enhancement of long-term potentiation by the calcium channel agonist Bayer K8644 in CA1 of the rat hippocampus in vitro
Neuroscience Letters, 80 (1987) 351 355
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 04836
Enhancement of long-term potentiation by the calcium channel agonist Bayer K8644 in C A 1 of the rat hippocampus in vitro Declan M u l k e e n l, R o g e r A n w y l 1 a n d Michael J. R o w a n 2 Departments of lPhysiology and :Pharmacology, Trinity College, Dublin (Ireland) (Received 2 March 1987; Revised version received 3 June 1987; Accepted 5 June 1987)
Key words': Long-term potentiation; BAY K8644; Hippocampal slice; CA~ The effect of the Ca agonist BAY K8644 was studied on long-term potentiation (LTP) of extracellular excitatory postsynaptic potentials in the stratum radiatum of CA~ of the hippocampus in vitro. LTP was evoked by brief trains of high-frequency stimulation applied to the stratum radiatum of CA~. 0.5% Ethanol, the vehicle used to dissolve BAY K8644, reduced LTP from 43% to 15%. An amount of 15/IM BAY K8644~ in 0.5% ethanol, enhanced LTP from 13% in the ethanol control to 57%. The Ca channel antagonist verapamil did not alter control LTP, but did inhibit the potentiating action of BAY K8644 on LTP. It is postulated that the enhancement of LTP by BAY K8644 may occur through enhancement of Ca influx through voltage-dependent Ca channels.
Brief high-frequency (50 250 Hz) stimulation of synaptic pathways in the hippocampus produces prolonged enhancement of postsynaptic responses to afferent fibre stimulation. This is usually termed long-term potentiation (LTP) [2, 17]. The production of LTP is known to depend on neuronal calcium entry, for experimentally reduced extracellular Ca or elevated Mg, which reduces Ca entry into neurones, antagonises LTP [5]. LTP may also be produced without high-frequency stimulation by briefly (4-7 min) elevating Ca in the perfusion media [20]. The Ca responsible for initiating LTP could enter the neurons via voltage-sensitive Ca channels [13] or via synaptic N-methyl-D-aspartate (NMDA) receptor channels [8]. The present experiments were designed to investigate whether Ca entry through voltage-dependent Ca channels can actually enhance LTP. This was carried out by using the dihydropyridine BAY K8644 which is known to increase Ca entry through voltage-dependent Ca channels. H ippocampal slices were prepared from 130 to 210 g Wistar rats as described previously [1] and maintained at 33 34°C in a medium containing (in mM): NaCI 120, KC1 2.5, CaCI2 2.0, MgSO4 !.0 or 2.0, NaHCO3 26, NaH2PO4 1.3, glucose 10. BAY K8644 was dissolved in ethanol before use, and diluted to 15 /lM in the medium. Correspondence." R. Anwyl, Department of Physiology, Trinity College, Dublin 2, Ireland.
352 In s o m e e x p e r i m e n t s v e r a p a m i l was p r e - a p p l i e d for 30 min before tetanic stimulation. S t i m u l a t i n g a n d r e c o r d i n g electrodes were glass m i c r o e l e c t r o d e s filled with 200 m M N a C I a n d having a resistance o f 1 M£2. T h e S c h a f f e r - c o l l a t e r a l p a t h w a y was stimulated, a n d field e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s (EPSPs) r e c o r d e d extracellularly from the s t a t u m r a d i a t u m . Slices s h o w i n g m u l t i p l e s p i k i n g in p y r a m i d a l layer recordings, o r which gave weak responses to stimuli, ( m a x i m a l EPSPs less t h a n twice the nerve volley a m p l i t u d e ) were excluded. T h e m a g n i t u d e o f c u r r e n t s t i m u l a t i o n was a d j u s t e d to give a c o n t r o l E P S P o f a m p l i t u d e 3 0 0 - 4 0 0 / t V at a frequency o f stimulation o f 0.03 Hz. L T P was i n d u c e d by a series o f 20 trains at 2 sec intervals, each train being 9 stimuli at 200 H z at a s t i m u l a t i o n c u r r e n t sufficient to p r o d u c e an E P S P o f a m p l i t u d e 800 # V at 0.03 Hz. The increase in L T P was expressed as the percentage increase in a 300/zV a m p l i t u d e EPSP. A m p l i t u d e , r a t h e r t h a n slope, o f the E P S P was m e a s u r e d as the r e l a t i o n s h i p between the slope a n d a m p l i t u d e o f the E P S P was linear up to a 0.8 mV E P S P a m p l i t u d e . T e t a n i c s t i m u l a t i o n o f the s t r a t u m r a d i a t u m c a u s e d an increase in the a m p l i t u d e o f the EPSPs following the tetanus. A s h o r t - l a s t i n g increase o f the EPSPs, p o s t - t e t a nic p o t e n t i a t i o n , o f 2 0 4 0 % m a g n i t u d e , declined within 5 10 min to a s t e a d y - s t a t e increase o v e r the c o n t r o l . This L T P lasted for over 45 min in the present e x p e r i m e n t s a n d was r o u t i n e l y m e a s u r e d at 20 rain p o s t - t e t a n u s . T h e L T P v a r i e d between 24% a n d 76% a n d a v e r a g e d 43% in c o n t r o l slices. In the presence o f 0.5% ethanol, which was used as the vehicle for B A Y K8644, L T P was significantly reduced, a v e r a g i n g 15% in the first g r o u p o f e x p e r i m e n t s ( P < 0.05, M a n n - W h i t n e y ) (Table I). T h e C a channel a g o n i s t d i h y d r o p y r i d i n e B A Y K8644 ( 1 5 / t M ) was f o u n d to p o t e n -
TABLE I EFFECTS OF ETHANOL AND BAY K8644 ON LONG-TERM POTENTIATION (LTP) Means & Ranges are the increases in LTP expressed as the percentage increase in the EPSP amplitude. *Significantly different from preceding group P<0.05 Mann-Whitney test. **Significantly different from preceding group P<0.01 Mann Whitney test. In each table, 3 of each group's slices were tested in I mM Mg2+ solution, and the remainder of both groups in 2 mM Mg 24 . Group
n
Mean %
The inhibitory effect of ethanol (86 mM) on LTP Control 7 42.5 Ethanol (86 mM)* 7 15 The excitatory effect of BAY K8644 on LTP Ethanol (86 mM) 9 Ethanol (86 mm) + 9 BAY K8644 (15/tM)**
12.9 57.4
Median %
Range %
25 10
1~76 4-28
7 43
The lack of effect of BAY K8644 on LTP in the presence of verapamil Ethanol+verapamil (10/tM) 5 27.2 33 Ethanol + BAY K8644 + 5 28.2 24 Verapamil (10/tM)
0 28 0--139
8 52 13 53
353
tiate LTP. Thus in BAY K8644, with 0.5% ethanol as vehicle, LTP averaged 57% (P < 0.01, Mann Whitney) (Table I, Fig. 1). The organic Ca channel antagonist verapamil inhibited the potentiating effects of BAY K 8644. Verapamil (10/~M) was firstly K8644+ETOH
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Fig. 1. a: graph showing the increase in the amplitude of the field EPSPs following a tetanus (marked by arrow) of high-frequency trains of stimuli (20 trains at 2-s intervals, each train consists of 9 stimuli at 200 Hz) in control media containing 0.5% ethanol (ETOH) ( O ) and in 15/~M BAY K8644 (IB) dissolved in ETOH. Data from 2 separate slices from the same rat. Note the large increase in long-term potentiation in the presence of BAY K8644, from 35% in control to 131% in BAY K8644. b: records of individual field EPSPs; (i) before (top trace) and after (bottom trace) high frequency stimulation in control media containing 0.5% ethanol, (ii) before (top trace) and after (bottom trace) high-frequency stimulation in the presence of 15 ILM K8644. Negativity shown in downwards deflection.
354
applied solely with 0.5% ethanol. LTP measured 27%, a value not significantly different from the vehicle control. Moreover, in 10/~M verapamil applied together with BAY K8644 and ethanol, LTP measured 28%, which was also not significantly different from the vehicle control (Table I). It is postulated that the increase in LTP by BAY K8644 is caused by an enhanced Ca influx into a pre- or postsynaptic site. BAY K8644 is known to enhance voltagedependent Ca currents in a variety of excitable cells [4, 9, 10]. In myocytes, BAY K8644 increased the open duration time of voltage-dependent Ca channels during depolarization, and thereby increased the maximum amplitude of whole-cell Ca currents [9, 10, 15]. BAY K8644 mainly enhances the open time duration of the L type of Ca channels, which generate the persistent non-inactivating Ca currents, in myocytes [15] and in hippocampal pyramidal neurons, although a transient current is also somewhat increased in the latter [4]. There are two proposed ways in which the Ca responsible for LTP enters the neurons during high-frequency stimulation, the voltage-dependent Ca channels [13] and NMDA receptor channels, which have a high Ca permeability [7, 8, 12]. The results of the present study showing enhancement of LTP by BAY K8644 does support the theory that Ca entry through voltage-dependent channels does initiate part of the normal tetanically produced LTP. BAY K8644 does not cause opening of Ca channels, but only prolongs opening of channels that have been opened by depolarisation. Therefore voltage-dependent Ca channels are likely to be participating in normal LTP in CA1. Ca entry is also likely to occur through activated NMDA receptors during tetanically evoked LTP through, as these receptors have a high permeability to Ca [12]. However, such Ca influx through the NMDA receptor is not always obligatory for initiating LTP as it has recently been shown that LTP in the stratum lucidum of CA3 is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate [6]. The increased LTP in CA1 mediated by 4-aminopyridine also suggested that Ca influx through voltage-dependent Ca channels participate in generating LTP [11]. The lack of effect of verapamil in blocking LTP in control media supports previous studies demonstrating the absence of an inhibitory effect of verapamil, flunarizine and nitrendipine on LTP [19]. Little conclusion can be made about this negative result though since these organic calcium channel antagonists do not inhibit Ca spikes in hippocampal pyramidal neurons [19]. The block of the BAY K8644 enhancement of LTP by verapamil in the present study does demonstrate though that Ca channels in the hippocampal pyramidal cells do have binding sites for these organic antagonists. However, rather than causing block of channel openings, there is an inhibitory action at the BAY K8644 binding site. The finding that ethanol inhibits LTP is in agreement with the recent studies showing that 100 mM ethanol antagonised LTP produced by tetanic stimulation or by a short period of exposure to high Ca [18]. Ethanol at similar concentrations reduced Ca influx through voltage-dependent Ca channels in a variety of excitable cells [14]. Such blockage of LTP by ethanol may be further evidence that the Ca influx producing LTP during tetanic stimulation does occur through voltage-dependent Ca channels.
355
Supported by the Medical Research Council of Ireland. 1 Anwyl, R. and Rowan, M.J., Frequency dependent block of field potentials in the rat hippocampal slice caused by tricyclic antidepressants, Br. J. Pharmacol., 86 (1985) 201 208. 2 Bliss, T.V.P. and Lomo, T., Long lasting potentiation of synaptic transmission in the dentate area ot" the anaesthetised rabbit following stimulation of the perforant path, J. Physiol. (London), 232 (1979) 331 356. 3 Collingridge, G.L., Kehl, S.J. and McLennen, H., Excitatory amino acids in synaptic transmission in the Schaffer collateral-commissural pathway of the rat hippocampus, J. Physiol. (London), 334 (1983) 33 46. 4 Docherty, R.J. and Brown, D.A., Interaction of 1,4-dihydropyridine with somatic Ca currents in hippocampal CA~ neurones in the guinea pig in vitro, Neurosci. Lett., 70 (1986) 110-115, 5 Dunwiddie, T.V. and Lynch, G., The relationship between extracellular calcium concentration and the induction of hippocampal long-term potentiation, Brain Res., 169 (1979) 103- l 10. 6 Harris, E.W. and Cotman, C.W., Long-term potentiation of guinea pig mossy fibre responses is not blocked by N-methyl-D-aspartate antagonists, Neurosci. Lett., 70 (1986) 132-137. 7 Harris, E.W., Ganong, A.H. and Cotman, C.W., Long term potentiation in the hippocampus involves activation of N-methyl-t~-aspartate receptors, Brain Res., 323 (1984) 132-137. 8 Herron, E.C., Lester, R.A.J., Coan, E.J. and Collingridge, G.L., Frequency involvement of NMDA reception in the hippocampus: a novel mechanism, Nature (London), 322 (1986) 265-267. 9 Hess, P. and Tsien, R.W., Mechanism of ion permeation through Ca channels, Nature (London), 309 (1984) 453456. 10 Kokubun, S. and Reuter, H., Dihydropyridine derivatives prolong the open state of Ca channels in cultured cardiac cells, Proc. Natl. Acad. Sci. USA, 81 (1984) 482~4827. 11 Lee, W.L., Anwyl, R. and Rowan, M.J., 4-Aminopyridine mediated increase in long term potentiation in CA~ of the rat hippocampus, Neurosci. Lett., 70 (1986) I06-109. 12 MacDermott, A.B., Mayer, M.L., Westbrook, G.L., Smith, S.J. and Barker, L.J., NMDA-receptor activation increases cytoplasmic calcium concentration in cultured spinal cord neurones, Nature (London), 321 (1986) 519 522. 13 Malinow, R. and Miller, J.P., Postsynaptic hyperpolarisation during conditioning reversibly blocks induction of long-term potentiation, Nature (London), 320 (1986) 529 530. 14 Messing, R.O., Carpenter, C.L., Diamond, I. and Greenberg, D.A., Ethanol regulates calcium channels in clinical neural cells, Proc. Natl. Acad. Sci. USA, 83 (1986) 6213 6215. 15 Nowycky, M.C., Fox, A.P. and Tsien, R.W., Long-opening mode of gating of neuronal calcium channels and its promotion by dihydropyridine calcium agonist BAY K8644, Proc. Natl. Acad. Sci. USA, 82 (1985) 2178 2182. 16 Sastry, B.R., Goh, J.W. and Pandanaboina, M.M., Verapamil counteracts the masking of long-lasting potentiation of hippocampal population spike produced by 2-amino-5-phosphorovalerate, Life Sci., 34 (1984) 323 329. 17 Schwartzkroin, P.A. and Wester, K., Long lasting facilitation o f a synaptic potential following tetanization in the in vitro hippocampal slice, Brain Res., 89 (1975) 107 119. 18 Sinclair, J.G. and Lo, G.F., Ethanol blocks tetanic and calcium induced long-term potentiation in the hippocampal slice, Gen. Pharmacol., 17 (1986) 231-233. 19 Taube, J.S. and Schwartzkroin, P.A., Ineffectiveness of organic calcium channel blockers in antagonizing long-term potentiation, Brain Res., 379 (1986) 275 285. 20 Turner, R.W., Bainbridge, K.G. and Miller, J.J., Calcium-induced long-term potentiation in the hippocampus, Neuroscience, 7 (1982) 1411 1416.
351
Elsevier Scientific Publishers Ireland Ltd.
NSL 04836
Enhancement of long-term potentiation by the calcium channel agonist Bayer K8644 in C A 1 of the rat hippocampus in vitro Declan M u l k e e n l, R o g e r A n w y l 1 a n d Michael J. R o w a n 2 Departments of lPhysiology and :Pharmacology, Trinity College, Dublin (Ireland) (Received 2 March 1987; Revised version received 3 June 1987; Accepted 5 June 1987)
Key words': Long-term potentiation; BAY K8644; Hippocampal slice; CA~ The effect of the Ca agonist BAY K8644 was studied on long-term potentiation (LTP) of extracellular excitatory postsynaptic potentials in the stratum radiatum of CA~ of the hippocampus in vitro. LTP was evoked by brief trains of high-frequency stimulation applied to the stratum radiatum of CA~. 0.5% Ethanol, the vehicle used to dissolve BAY K8644, reduced LTP from 43% to 15%. An amount of 15/IM BAY K8644~ in 0.5% ethanol, enhanced LTP from 13% in the ethanol control to 57%. The Ca channel antagonist verapamil did not alter control LTP, but did inhibit the potentiating action of BAY K8644 on LTP. It is postulated that the enhancement of LTP by BAY K8644 may occur through enhancement of Ca influx through voltage-dependent Ca channels.
Brief high-frequency (50 250 Hz) stimulation of synaptic pathways in the hippocampus produces prolonged enhancement of postsynaptic responses to afferent fibre stimulation. This is usually termed long-term potentiation (LTP) [2, 17]. The production of LTP is known to depend on neuronal calcium entry, for experimentally reduced extracellular Ca or elevated Mg, which reduces Ca entry into neurones, antagonises LTP [5]. LTP may also be produced without high-frequency stimulation by briefly (4-7 min) elevating Ca in the perfusion media [20]. The Ca responsible for initiating LTP could enter the neurons via voltage-sensitive Ca channels [13] or via synaptic N-methyl-D-aspartate (NMDA) receptor channels [8]. The present experiments were designed to investigate whether Ca entry through voltage-dependent Ca channels can actually enhance LTP. This was carried out by using the dihydropyridine BAY K8644 which is known to increase Ca entry through voltage-dependent Ca channels. H ippocampal slices were prepared from 130 to 210 g Wistar rats as described previously [1] and maintained at 33 34°C in a medium containing (in mM): NaCI 120, KC1 2.5, CaCI2 2.0, MgSO4 !.0 or 2.0, NaHCO3 26, NaH2PO4 1.3, glucose 10. BAY K8644 was dissolved in ethanol before use, and diluted to 15 /lM in the medium. Correspondence." R. Anwyl, Department of Physiology, Trinity College, Dublin 2, Ireland.
352 In s o m e e x p e r i m e n t s v e r a p a m i l was p r e - a p p l i e d for 30 min before tetanic stimulation. S t i m u l a t i n g a n d r e c o r d i n g electrodes were glass m i c r o e l e c t r o d e s filled with 200 m M N a C I a n d having a resistance o f 1 M£2. T h e S c h a f f e r - c o l l a t e r a l p a t h w a y was stimulated, a n d field e x c i t a t o r y p o s t s y n a p t i c p o t e n t i a l s (EPSPs) r e c o r d e d extracellularly from the s t a t u m r a d i a t u m . Slices s h o w i n g m u l t i p l e s p i k i n g in p y r a m i d a l layer recordings, o r which gave weak responses to stimuli, ( m a x i m a l EPSPs less t h a n twice the nerve volley a m p l i t u d e ) were excluded. T h e m a g n i t u d e o f c u r r e n t s t i m u l a t i o n was a d j u s t e d to give a c o n t r o l E P S P o f a m p l i t u d e 3 0 0 - 4 0 0 / t V at a frequency o f stimulation o f 0.03 Hz. L T P was i n d u c e d by a series o f 20 trains at 2 sec intervals, each train being 9 stimuli at 200 H z at a s t i m u l a t i o n c u r r e n t sufficient to p r o d u c e an E P S P o f a m p l i t u d e 800 # V at 0.03 Hz. The increase in L T P was expressed as the percentage increase in a 300/zV a m p l i t u d e EPSP. A m p l i t u d e , r a t h e r t h a n slope, o f the E P S P was m e a s u r e d as the r e l a t i o n s h i p between the slope a n d a m p l i t u d e o f the E P S P was linear up to a 0.8 mV E P S P a m p l i t u d e . T e t a n i c s t i m u l a t i o n o f the s t r a t u m r a d i a t u m c a u s e d an increase in the a m p l i t u d e o f the EPSPs following the tetanus. A s h o r t - l a s t i n g increase o f the EPSPs, p o s t - t e t a nic p o t e n t i a t i o n , o f 2 0 4 0 % m a g n i t u d e , declined within 5 10 min to a s t e a d y - s t a t e increase o v e r the c o n t r o l . This L T P lasted for over 45 min in the present e x p e r i m e n t s a n d was r o u t i n e l y m e a s u r e d at 20 rain p o s t - t e t a n u s . T h e L T P v a r i e d between 24% a n d 76% a n d a v e r a g e d 43% in c o n t r o l slices. In the presence o f 0.5% ethanol, which was used as the vehicle for B A Y K8644, L T P was significantly reduced, a v e r a g i n g 15% in the first g r o u p o f e x p e r i m e n t s ( P < 0.05, M a n n - W h i t n e y ) (Table I). T h e C a channel a g o n i s t d i h y d r o p y r i d i n e B A Y K8644 ( 1 5 / t M ) was f o u n d to p o t e n -
TABLE I EFFECTS OF ETHANOL AND BAY K8644 ON LONG-TERM POTENTIATION (LTP) Means & Ranges are the increases in LTP expressed as the percentage increase in the EPSP amplitude. *Significantly different from preceding group P<0.05 Mann-Whitney test. **Significantly different from preceding group P<0.01 Mann Whitney test. In each table, 3 of each group's slices were tested in I mM Mg2+ solution, and the remainder of both groups in 2 mM Mg 24 . Group
n
Mean %
The inhibitory effect of ethanol (86 mM) on LTP Control 7 42.5 Ethanol (86 mM)* 7 15 The excitatory effect of BAY K8644 on LTP Ethanol (86 mM) 9 Ethanol (86 mm) + 9 BAY K8644 (15/tM)**
12.9 57.4
Median %
Range %
25 10
1~76 4-28
7 43
The lack of effect of BAY K8644 on LTP in the presence of verapamil Ethanol+verapamil (10/tM) 5 27.2 33 Ethanol + BAY K8644 + 5 28.2 24 Verapamil (10/tM)
0 28 0--139
8 52 13 53
353
tiate LTP. Thus in BAY K8644, with 0.5% ethanol as vehicle, LTP averaged 57% (P < 0.01, Mann Whitney) (Table I, Fig. 1). The organic Ca channel antagonist verapamil inhibited the potentiating effects of BAY K 8644. Verapamil (10/~M) was firstly K8644+ETOH
BAY
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~
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%,
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a. ffl a. .1
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0 I
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Fig. 1. a: graph showing the increase in the amplitude of the field EPSPs following a tetanus (marked by arrow) of high-frequency trains of stimuli (20 trains at 2-s intervals, each train consists of 9 stimuli at 200 Hz) in control media containing 0.5% ethanol (ETOH) ( O ) and in 15/~M BAY K8644 (IB) dissolved in ETOH. Data from 2 separate slices from the same rat. Note the large increase in long-term potentiation in the presence of BAY K8644, from 35% in control to 131% in BAY K8644. b: records of individual field EPSPs; (i) before (top trace) and after (bottom trace) high frequency stimulation in control media containing 0.5% ethanol, (ii) before (top trace) and after (bottom trace) high-frequency stimulation in the presence of 15 ILM K8644. Negativity shown in downwards deflection.
354
applied solely with 0.5% ethanol. LTP measured 27%, a value not significantly different from the vehicle control. Moreover, in 10/~M verapamil applied together with BAY K8644 and ethanol, LTP measured 28%, which was also not significantly different from the vehicle control (Table I). It is postulated that the increase in LTP by BAY K8644 is caused by an enhanced Ca influx into a pre- or postsynaptic site. BAY K8644 is known to enhance voltagedependent Ca currents in a variety of excitable cells [4, 9, 10]. In myocytes, BAY K8644 increased the open duration time of voltage-dependent Ca channels during depolarization, and thereby increased the maximum amplitude of whole-cell Ca currents [9, 10, 15]. BAY K8644 mainly enhances the open time duration of the L type of Ca channels, which generate the persistent non-inactivating Ca currents, in myocytes [15] and in hippocampal pyramidal neurons, although a transient current is also somewhat increased in the latter [4]. There are two proposed ways in which the Ca responsible for LTP enters the neurons during high-frequency stimulation, the voltage-dependent Ca channels [13] and NMDA receptor channels, which have a high Ca permeability [7, 8, 12]. The results of the present study showing enhancement of LTP by BAY K8644 does support the theory that Ca entry through voltage-dependent channels does initiate part of the normal tetanically produced LTP. BAY K8644 does not cause opening of Ca channels, but only prolongs opening of channels that have been opened by depolarisation. Therefore voltage-dependent Ca channels are likely to be participating in normal LTP in CA1. Ca entry is also likely to occur through activated NMDA receptors during tetanically evoked LTP through, as these receptors have a high permeability to Ca [12]. However, such Ca influx through the NMDA receptor is not always obligatory for initiating LTP as it has recently been shown that LTP in the stratum lucidum of CA3 is not blocked by the NMDA receptor antagonist 2-amino-5-phosphonovalerate [6]. The increased LTP in CA1 mediated by 4-aminopyridine also suggested that Ca influx through voltage-dependent Ca channels participate in generating LTP [11]. The lack of effect of verapamil in blocking LTP in control media supports previous studies demonstrating the absence of an inhibitory effect of verapamil, flunarizine and nitrendipine on LTP [19]. Little conclusion can be made about this negative result though since these organic calcium channel antagonists do not inhibit Ca spikes in hippocampal pyramidal neurons [19]. The block of the BAY K8644 enhancement of LTP by verapamil in the present study does demonstrate though that Ca channels in the hippocampal pyramidal cells do have binding sites for these organic antagonists. However, rather than causing block of channel openings, there is an inhibitory action at the BAY K8644 binding site. The finding that ethanol inhibits LTP is in agreement with the recent studies showing that 100 mM ethanol antagonised LTP produced by tetanic stimulation or by a short period of exposure to high Ca [18]. Ethanol at similar concentrations reduced Ca influx through voltage-dependent Ca channels in a variety of excitable cells [14]. Such blockage of LTP by ethanol may be further evidence that the Ca influx producing LTP during tetanic stimulation does occur through voltage-dependent Ca channels.
355
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